Currently, methods of staining biological samples to obtain both morphological and molecular details are based on two types of illumination, fluorescence illumination and brightfield illumination. Brightfield illumination utilizes stains specifically developed to provide morphological details, such as cell type or disease state. However, these morphological stains do not provide information at the molecular level. To remedy this, fluorescent illumination methods utilize stains specifically developed to provide information at the molecular level. For example, fluorescence stains are useful for examining alterations in a cell at the deoxyribonucleic acid (DNA) level. However, fluorescence stains do not provide morphological information required to identify cell type or disease state. Therefore, a combination counterstain used to stain a biological sample that would allow examination by both fluorescence illumination and brightfield illumination on the same microscope slide would be very useful.
Typically, fluorescence stains and brightfield stains are not combined on the same microscope slide because the two methods are incompatible with each other. When a fluorescence stain is examined under brightfield illumination no staining is observed. Likewise, when brightfield stains are examined by fluorescence illumination either no staining is observed or high background staining, without specific staining is observed.
Current attempts to combine these two staining methods have been ineffective, because the counterstains used for brightfield illumination exhibit unwanted characteristics such as quenching, background, and interference when examined by fluorescence. The usefulness of combining these two staining methods has been recognized and certain work-around methods have been developed.
The most commonly used work-around method involves the preparation of two microscope slides from the same biological sample. From a typical biological sample, multiple microscope slides can be prepared. Typically, the biological sample is cut into thin tissue slices of approximately 4μ, and each slide is affixed to a microscope slide. When these 4μ sections are kept in order and sequentially laid onto a series of microscope slides, the slides are said to be serial sections meaning that the slides are ordered in the same way that the tissue was ordered in the biological sample. The implication is that tissues mounted on serial sections do not vary greatly from each other. They vary only by 4μ, which is less than the diameter of a single cell. Therefore, serial sections are considered to be nearly identical to each other. Thus, it is possible to stain a first section on a first slide with a morphological stain and a serial section on a second microscope slide with a molecular stain. The first slide can be examined by brightfield illumination and the cell type or disease state can be identified. The second slide is then examined by fluorescence illumination and molecular alterations are identified. The microscopist will then mentally reconstruct the two images to determine which cells are exhibiting the altered molecular state. However, the mental process of reconstructing two images can be difficult and result in error. Furthermore, the requirements of staining two different slides make this method more time consuming and expensive compared with staining a single slide using a combined stain.
Therefore, it would be advantageous to be able to combine both brightfield and fluorescence stains within a single slide. By combining the information obtained from both stains on a single slide the investigator can determine both the cell type and specific molecular alterations associated with that cell. For example, a brightfield stain provides the investigator with critical morphological information such that the specific cell type (for example, cancerous) can be identified, and fluorescence examination allows the investigator to observe specific molecular alterations (for example, a molecular marker) within the cell.